Genotype-phenotype correlation of TGFBI corneal dystrophies in Polish patients.

PURPOSE
To analyze genotype-phenotype correlation in patients originating from Polish population with the transforming growth factor beta induced (TGFBI) corneal dystrophies.


METHODS
Sixty affected and 31 unaffected individuals from 15 unrelated Polish families were included in the study. The clinical diagnosis was based on the slit-lamp exam, 1310 nm time domain and 1310 nm swept source spectral domain optical coherence tomography (OCT). Histopathologic analysis was performed on 10 available corneal buttons. Exons of the TGFBI gene were screened for mutations with polymerase chain reaction (PCR) and direct DNA sequencing.


RESULTS
We found the lattice phenotype dominant compared to the granular one in the Polish population (41:16 patients; lattice:granular). We identified five distinct mutations responsible for TGFBI corneal dystrophies (R124R, R124H, R555W, R555Q, and H626R). There was a strong genotype-phenotype correlation in the case of R124R and R555W mutations, while there was a distinct phenotypic heterogeneity in the case of the H626R mutation. OCT analysis revealed that the reflectivity, location and pattern of the corneal deposits were different among the TGFBI corneal dystrophies. The advantage of spectral swept source OCT over time-domain OCT scans is a more distinct visualization of the Bowman's layer area and deposits located under the epithelium.


CONCLUSIONS
This study underlines the role of comprehensive phenotype-genotype analysis in TGFBI corneal dystrophies, describes for the first time the TGFBI mutation spectrum in a Polish population and reveals phenotypic heterogeneity in the case of the H626R mutation.

More than 50 distinct disease-causing mutations have been identified in the TGFBI gene [2]. Different populationwide studies revealed two mutational hot spots at codons R124 and R555 [3][4][5]. Researchers have established that TGFBI corneal dystrophies are inherited with complete penetrance and dominance and certain TGFBI mutations are correlated with a specific phenotype. Researchers have also established that the R124C mutation is associated with lattice corneal dystrophy type I, the R124L mutation is associated with Reis-Bucklers corneal dystrophy, the R555W mutation is associated with granular corneal dystrophy type I, the R555Q mutation is associated with Thiel-Behnke corneal dystrophy, whereas granular corneal dystrophy type II is associated with the R124H mutation. Later studies reported cases of increased severity of deposits, multiple recurrences and earlier onset for patients bearing homozygous mutations suggesting a semidominant inheritance pattern [6][7][8][9][10][11]. Cases of reduced penetrance have also been reported [12,13]. Several reports underlined the phenotypic and mutational heterogeneity according to rare mutations and, in recent years, to common TGFBI mutations [14][15][16][17][18][19][20].
Although molecular studies in differential diagnosis of TGFBI dystrophies have been used increasingly in clinical practice during recent years, phenotype analysis and histopathologic examination remain the "gold standard" for determining the type of corneal dystrophy. Additionally, in recent years, corneal imaging techniques have been developed that allow in vivo corneal morphology and morphometry assessment to analyze the disease progression and determine the method of treatment. Confocal microscopy, invented in 1955 and developed in the nineties, has been used for highresolution imaging and provides en face images of the corneal layers [21]. The IC3D classification system contains a description of confocal microscopy images of all corneal dystrophies. Optical coherence tomography (OCT) is a highspeed, high-resolution, non-contact optical imaging technique introduced in 1991 for cross-sectional imaging in biologic systems [22]. Anterior eye segment imaging with the 830 nm light wavelength OCT was demonstrated in 1994 [23]. Introduction of transscleral anterior eye segment imaging was achieved by changing the light wavelength from 830 nm to 1310 nm [24]. In recent years, OCT technology has been revolutionized by the development of spectral domain technology, which provides an increased signal-to-noise ratio and increased robustness compared with time domain OCT [25]. OCT is proven to provide reliable anterior eye segment parameters measurements characterized by good repeatability and reproducibility [26].
We report, for the first time, the TGFBI mutation spectrum and genotype-phenotype correlation integrating the slit-lamp exam, corneal morphology assessment with two OCT systems, histological exam and genetic analysis of patients encompassing a population in Poland.

METHODS
The study was approved by the Ethics Committee of the Medical University of Silesia, Katowice, Poland (SUM NN-6501-175/I/07). All patients had to sign informed consent forms before undergoing any study procedure. A family was included in the study only if at least two family members had been diagnosed with TGFBI corneal dystrophy.
Patients: Sixty affected and 31 unaffected individuals (50 women and 41 men) from 15 unrelated Polish families were included in the study (family F1-F15). The mean age of the patients was 46 years (±19 years). The mean number of examined patients was 5 per family (ranging from 3 to 11 patients). Seventeen patients had undergone a keratoplasty procedure in the past, while 9 patients underwent keratoplasty while this study was performed.
Phenotype analysis: A detailed family history was collected, and pedigree charts were constructed for all patients. We analyzed the age at onset of symptoms and type of symptoms reported by patients. The clinical examination consisted of visual acuity, slit-lamp biomicroscopy with photography, time domain and spectral domain optical coherence tomography.
We analyzed corneal change patterns, location, symmetry and progression with age. In addition, heterogeneity of corneal morphology among patients with the same TGFBI mutation was assessed.
Histologic examination: The 10 available corneal buttons from 9 patients (6 without the Descemet membrane and posterior corneal stroma after deep anterior lamellar keratoplasty and 4 whole corneal buttons after penetrating keratoplasty) were processed by standard methods involving sectioning of the tissue samples. Corneal sections were stained with hematoxylin and eosin (HE), periodic acid-Schiff (PAS), Alcian blue, Masson's trichrome, and Congo red and further analyzed with light microscopy for the presence of amyloid and/or hyaline deposits. The presence of amyloids was confirmed by birefringence when viewed under polarized light.
DNA collection, isolation, amplification and sequencing: DNA was isolated from dry blood samples collected on FTA ® cards (Whatmann, Maidstone, UK). For DNA isolation, a 2.0-mm-diameter disc was punched and collected into a sterile Eppendorf tube. DNA was isolated by using the lysis and neutralization solutions from REDExtract-N-Amp™ Blood PCR Kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's protocol. Exons were amplified by polymerase chain reaction (PCR) with the primers published by Munier et al. [3] ( Table 1). The PCR product was purified and directly submitted for DNA sequencing. In case of 15 probands, we started the analysis with screening exon 4 and exon 12 of the TGFBI gene, where hot spot sites for mutations have been previously reported [2][3][4][5]. If no mutation was revealed, we screened TGFBI exon by exon until the mutation was defined. After that, in case of other family members, we analyzed the exons for which the mutation was defined for the proband.
Direct sequencing was performed using the BigDye Terminator v3.1 (Applied Biosystems; Life Technologies, Foster City, CA). A 3730xl DNA Analyzer (Applied Biosystems) was used to collect the sequence data. Nucleotide sequences were compared with the published sequence of TGFBI (GenBank NM_000358) to analyze the sequence changes.
The disease showed an autosomal dominant inheritance pattern in all families as revealed by pedigree analysis. The age of onset varied between different corneal dystrophies. In the case of patients with TBCD, LCDI, and GCDI and II, the onset of the disease was in the first and second decades of life. In patients with LCD variants, the onset was delayed to the fourth and fifth decades of life. Patients from all studied families reported recurrent erosion and visual impairment during the course of the disease. Results of genetic analysis with comparison to corneal phenotypes are summarized in Table 2. Optical coherence tomography: All corneal changes were hyperreflective in the time domain and spectral OCT scans, but the level of increased reflectivity was different for different types of corneal dystrophies and increased in the following order: lattice corneal dystrophy, Thiel-Behnke corneal dystrophy, granular corneal dystrophy type I and granular corneal dystrophy type II. The opacities also were different in shape and pattern: from diffuse areas of increased reflectivity in lattice corneal dystrophy type I to focal, distinct and hyperreflective opacities in granular corneal dystrophies type I and II ( Figure 1B,C,E,F,H,I and Figure 2B,D,F).
When comparing images of time-domain and spectral domain OCT, we noticed that all changes revealed on spectral swept source OCT scans were also visible on time-domain

Exon
Sequence of forward and reverse primer Annealing temp (°C) Product size (bp) Based on data previously published by Munier et al. [3].   OCT. That makes both techniques useful for establishing the phenotype features of each TGFBI corneal dystrophy and monitoring the progression of the disease. The advantage of spectral swept source OCT over time-domain OCT scans is more distinct visualization of the Bowman's layer area and the deposits located under the epithelium ( Figure  1B,C,E,F,H,I).
Phenotypic heterogeneity: Patients with TGFBI mutations R124C and R555W were characterized by a homogenous phenotype. The corneal phenotype of patients with the R555W mutation (families F4, F10, and F11) was characterized by granular crumb-like deposits in the central cornea, with age becoming located deeper in the stroma. In OCT scans, focal distinct opacities of increased reflectivity corresponding to corneal deposits were visible ( Figure 1A-C). Two available corneal buttons revealed the interruption or local absence of Bowman's layer, irregularity of the epithelium in correspondence with the superficial granular deposits and multiple granular deposits in the corneal stroma ( Figure 3C,D). The corneal phenotype of patients with the R124C mutation (Families F1, F3, F5, F6, F12, F13, F14, and F15) was characterized by fragile, multiple lattice lines in the anterior to mid stroma in the central cornea with diffuse areas of increased reflectivity on corresponding OCT scans ( Figure   2A,B). Five available corneal buttons revealed deposits that stained positive with Congo red. Green birefringence was visible with a polarizing filter ( Figure 3A). Accompanying changes involved thinning of the epithelium and interruption of Bowman's layer.
In the case of patients with R124H and R555Q mutations, we could not assess interfamilial heterogeneity, because there was only 1 family of each type. The phenotype of the R555Q mutation (family F8) was characterized by a "honeycomb" pattern of corneal deposits in the anterior corneal part. The changes visible on the OCT scans were limited to the anterior corneal stroma and the area of Bowman's layer ( Figure 1G-I). Since no members of family F8 underwent keratoplasty, the corneal button for histologic analysis was not available. The corneal changes in family F9 with the R124H mutation involved granular-, star-and disc-shaped stromal opacities without lattice lines. The opacities were highly reflective on OCT scans ( Figure 1D-F). Histologic analysis revealed multiple granular deposits stained positively with Masson trichrome and a few sub Bowman's layer Congo red positive amyloid deposits ( Figure 3E,F).
Patients with the H626R mutation (Families F2 and F7) revealed interfamilial heterogeneity ( Figure 2C-F). Corneal changes were asymmetric in both families. On the slit-lamp exam, a few thin, fragile lattice lines were located centrally in patients from family F2, while the lines were thick, distinct, extended to limbus and accompanied by stromal haze in patients from the family F7. OCT scans revealed that corneal opacities were located deep in the stroma in both families, even with deformation of the posterior corneal surface in family F7 (Figure 2C-F). The examination of one corneal button of family F2 confirmed the deep location of Congo red positive deposits in the corneal stroma ( Figure 3B).

DISCUSSION
The development of molecular analysis has changed our understanding of corneal dystrophies. In 1997, Munier et al. [27] reported that mutation of one gene (TGFBI) caused dystrophies of different corneal layers. Consequently, this finding rendered classification based on corneal phenotype archaic, and it was replaced by the new IC3D system in 2008 [1].
In this first comprehensive report of TGFBI mutations covering a population in Poland, we found the lattice phenotype was dominant compared to the granular one (41:16 patients; lattice:granular). We identified five distinct mutations responsible for TGFBI corneal dystrophies (R124R, R124H, R555W, R555Q, and H626R). All those mutations were previously described. There was a strong genotype-phenotype correlation in the case of R124R (families F1, F3, F5, F6, F12, F13, F14, and F15) and R555W (families F4, F10, and F11) mutations, while there was a distinct phenotypic heterogeneity in the case of the H626R mutation (families F2 and F7). The mutation was first described by Stewart et al. [32] and Clair-Florent et al. [33] in 1999. Previous authors reported delayed disease onset and asymmetry of corneal changes [32][33][34][35][36]. The lattice lines were described as thicker and more distinct than in LCD type 1, which resembles the phenotype of our family F7. None of the previous authors revealed such deep deposits location that they caused the deformation of the posterior corneal surface as observed on OCT images of affected members from family F7 ( Figure 2F). However, previous reports were based on slitlamp and histopathologic findings, while in our study the deep corneal deposits affecting the posterior corneal surface were revealed exclusively based on OCT exams, not visible on the slit-lamp exam.
In our study, the phenotype of Avellino corneal dystrophy (family F9) differed from the classical form containing both granular and lattice deposits on the slit-lamp exam. In the case of family F9, no lattice lines were detectable on the slit-lamp and OCT exams ( Figure 1D-F). Such heterogeneity was previously reported [37]. In our case, the diagnosis of GCDII was defined by revealing subepithelial Congo red positive deposits ( Figure 3E,F) and confirming the presence of the R124H mutation. This underlines the need for precise phenotype analysis and TGFBI screening for definite diagnosis and classification of corneal dystrophies.
The current IC3D classification system contains the description of each corneal dystrophy based on slit-lamp, confocal microscopy, histopathology and transmission electron microscopy combined with the results of genetic analysis. In recent years, reports underlying the role of anterior segment optical coherence tomography in visualization of corneal morphology in corneal dystrophies have been published [38][39][40]. However, most papers were based on case reports of a single corneal dystrophy type, and the exam was performed by one OCT system. We confirmed the utility of time-domain and spectral OCT systems in establishing the characteristic features and the depth of corneal changes. Analyzing the depth of corneal deposits is one of the most important factors in choosing a treatment method. Performing an OCT exam is surely more detailed than the slit-lamp exam in assessing Descemet membrane involvement. OCT examination was already proven to be useful in optical coherence tomography-guided phototherapeutic keratectomy or in selection of lamellar versus penetrating keratoplasty as the treatment procedure of choice [41,42]. The main advantages of spectral versus time-domain OCT are the ability to visualize early corneal changes affecting the Bowman's layer area (Figure 1) and the possibility of creating threedimensional images. Compared to the slit-lamp examination, OCT provides detailed information about the disease progression based on documented analysis of corneal opacities' depth and extent. Compared to confocal microscopy, OCT acquires less detailed information with axial resolution to 5 µm, but provides information about the range of corneal opacities and measures corneal thickness. In contrast to histopathologic examination, OCT can be performed in the early stages of the disease, without the need for keratoplasty. The recent development of OCT shows that this technique is in continuous change. New OCT prototypes gain axial resolution of 2-3 µm, which is more than twice that of OCT devices already used [43]. By using these new OCT prototypes, we could improve non-contact corneal imaging in the future.
In conclusion, this study underlines the role of comprehensive phenotype-genotype analysis in TGFBI corneal dystrophies, describes the TGFBI mutation spectrum in a Polish population and reveals the phenotypic heterogeneity in the case of the H626R mutation.
This study also highlights the need for a combined corneal morphology and genetic assessment to establish the diagnosis of TGFBI corneal dystrophies.